U.S. patent number 11,065,348 [Application Number 15/320,177] was granted by the patent office on 2021-07-20 for apparatus and methods for making recombinant protein-stabilized monodisperse microbubbles.
This patent grant is currently assigned to The Trustees of the University of Pennsylvania. The grantee listed for this patent is THE TRUSTEES OF THE UNIVERSITY OF PENNSYLVANIA. Invention is credited to Francesco Angile, Daniel A. Hammer, Daeyeon Lee, Chandra M. Sehgal, Kevin Vargo.
United States Patent |
11,065,348 |
Lee , et al. |
July 20, 2021 |
**Please see images for:
( Certificate of Correction ) ** |
Apparatus and methods for making recombinant protein-stabilized
monodisperse microbubbles
Abstract
A microfluidic device for generating microbubbles includes a
substrate and a microfluidic channel embedded in the substrate. The
microfluidic channel includes a plurality of fluid inlets, at least
one bubble formation outlet having a nozzle with an adjustable
diameter, and a flow focusing junction in fluid communication with
the plurality of fluid inlets and the bubble formation outlet. A
method for mass producing monodisperse microbubbles with a
microfluidic device includes supplying a flow of dispersed phase
fluid into a first fluid inlet of a microfluidic channel, supplying
a flow of continuous phase fluid into a second fluid inlet of the
microfluidic channel, and adjusting a diameter of a nozzle to
obtain a plurality of monodisperse microbubbles having a specified
diameter.
Inventors: |
Lee; Daeyeon (Wynnewood,
PA), Angile; Francesco (Philadelphia, PA), Vargo;
Kevin (Philadelphia, PA), Hammer; Daniel A. (Villanova,
PA), Sehgal; Chandra M. (Wayne, PA) |
Applicant: |
Name |
City |
State |
Country |
Type |
THE TRUSTEES OF THE UNIVERSITY OF PENNSYLVANIA |
Philadelphia |
PA |
US |
|
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Assignee: |
The Trustees of the University of
Pennsylvania (Philadelphia, PA)
|
Family
ID: |
54936136 |
Appl.
No.: |
15/320,177 |
Filed: |
June 19, 2015 |
PCT
Filed: |
June 19, 2015 |
PCT No.: |
PCT/US2015/036678 |
371(c)(1),(2),(4) Date: |
December 19, 2016 |
PCT
Pub. No.: |
WO2015/196065 |
PCT
Pub. Date: |
December 23, 2015 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20170119911 A1 |
May 4, 2017 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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62014451 |
Jun 19, 2014 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
B01F
23/2323 (20220101); A61K 49/223 (20130101); C07K
14/415 (20130101); A61B 8/481 (20130101); B01F
33/3011 (20220101); A61K 38/00 (20130101); B01F
23/2373 (20220101); B01F 33/301 (20220101); B01F
2101/22 (20220101); B01F 2101/2202 (20220101); A61N
7/00 (20130101) |
Current International
Class: |
A61B
8/00 (20060101); A61K 49/22 (20060101); B01F
13/00 (20060101); B01F 3/04 (20060101); A61B
8/08 (20060101); C07K 14/415 (20060101); A61N
7/00 (20060101); A61K 38/00 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
Other References
Garstecki et al. (Appl. Phys. Lett. 2004, 85, 2649-2651). cited by
examiner .
Wang et al. (Biomicrofluidics 2013, 7, 014103-1 to 014103-12).
cited by examiner .
Choi et al. (Lab Chip 2010, 10, 456-461). cited by examiner .
Abate et al. (Appl. Phys. Lett. 92, 243509 (2008)). cited by
examiner .
International Preliminary Report on Patentability with Written
Opinion for International Application No. PCT/US2015/036678, dated
Dec. 29, 2016, 9 pages. cited by applicant .
International Search Report for International Application No.
PCT/US2015/036678, dated Sep. 21, 2015, 2 pages. cited by
applicant.
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Primary Examiner: Hartley; Michael G.
Assistant Examiner: Perreira; Melissa J
Attorney, Agent or Firm: BakerHostetler
Government Interests
GOVERNMENT RIGHTS
This invention was made with government support under grant numbers
DMR1120901 and DMR1309556 awarded by the National Science
Foundation. The government has certain rights in the invention.
Parent Case Text
CROSS-REFERENCE TO RELATED APPLICATIONS
This is the national phase of International Application No
PCT/US2015/036678, filed 19 Jun. 2015, which claims priority to
U.S. Provisional Application No. 62/014,051, filed 19 Jun. 2014.
The disclosure of each of these applications is incorporated herein
by reference in its entirety for all purposes.
Claims
What is claimed:
1. A microfluidic device for generating microbubbles, comprising:
(a) a substrate that defines a plane; (b) a microfluidic channel
system comprising a plurality of channels formed in the substrate
and extending in the plane of the substrate along a surface of the
substrate, the microfluidic channel system comprising: a plurality
of fluid inlets, at least one bubble formation outlet, the at least
one bubble formation outlet comprising a nozzle having an
adjustable diameter, the adjustable diameter of the nozzle
effecting control over the size of microbubbles exiting the nozzle,
and a flow focusing junction in fluid communication with the
plurality of fluid inlets and with the bubble formation outlet; and
(c) a dynamically actuatable valve that encircles the nozzle and is
adapted to inflate and dynamically constrict the nozzle of the
bubble formation outlet so as to change the adjustable diameter of
the nozzle, wherein the dynamically actuatable valve and the
microfluidic channel system lie in the plane of the substrate.
2. The microfluidic device of claim 1, wherein a first fluid inlet
of the plurality of fluid inlets comprises an inlet for a gas, and
a second fluid inlet of the plurality of fluid inlets comprises an
inlet for a liquid.
3. The microfluidic device of claim 1, wherein the first fluid
inlet and the second fluid discharge into the flow focusing
junction, and wherein the bubble formation outlet is disposed at
the flow focusing junction.
4. The microfluidic device of claim 1, wherein the dynamically
actuated valve is a fluid-actuated valve.
5. The microfluidic device of claim 1 comprising more than one
bubble formation outlet, wherein each of the one or more bubble
formation outlets comprises a respective nozzle having an
adjustable diameter.
6. The microfluidic device of claim 4, wherein the microfluidic
channel system and the valve define a direction of flow in the same
plane.
7. The microfluidic device of claim 6 wherein the substrate
comprises a polymer.
8. The microfluidic device of claim 7, wherein the polymer
comprises polydimethylsiloxane, a polyacrylamide, a polyacrylate, a
polymethacrylate or a mixture thereof.
9. The device of claim 1, further comprising at least one valve
control channel formed in the substrate and extending in the plane
of the substrate along a surface of the substrate, wherein the at
least one valve control channel is in fluid communication with the
valve, and wherein exertion of fluid in the at least one valve
control channel acts to inflate the dynamically actuated valve so
as to constrict the diameter of the nozzle.
Description
FIELD OF THE INVENTION
This invention relates to the field of microfluidics and, more
particularly, microbubbles as well as devices and processes for
producing microbubbles.
BACKGROUND OF THE INVENTION
Microbubbles are used as contrast enhancing agents in ultrasound
sonography and more recently have shown great potential as
theranostic agents that enable both diagnostics and therapy. The
use of microbubble contrast agents enables visualization of
microvasculature which cannot be seen directly with Doppler
ultrasound. The echogenicity of microbubbles coupled with their
physical interactions with acoustic energy can also be used for
triggered release of active agents, or for conversion of acoustic
energy to thermal energy to enable therapeutic applications. For
example, recent studies have shown that the insonation of
microbubbles with low-intensity ultrasound can lead to a localized
temperature increase, which in turn disrupts tumor vasculature
(also known as anti-vascular ultrasound therapy), enabling
minimally invasive procedure to disrupt cancerous tissues. These
properties of microbubbles make them ideal candidates for
theranostics; that is, the same microbubble agents can be used for
diagnostics and therapeutic applications.
Conventional production methods undesirably lead to highly
polydisperse microbubbles. Although some methods to fractionate
microbubbles to enhance the uniformity of size have been reported,
these techniques inevitably lead to loss of significant fraction of
bubbles. Similarly, while the generation of monodisperse bubbles
using microfluidic techniques has been reported, the size range of
microbubbles that can be generated from such devices is somewhat
limited.
Additionally, presently available microbubbles are typically
stabilized with materials that offer limited possibilities in
modifying the shell functionality for therapeutic applications.
These limitations compromise the effectiveness of microbubbles in
ultrasound imaging and novel theranostic approaches such as
targeted drug delivery and antivascular ultrasound therapy (AVUST).
For example, polydisperse microbubbles may drastically reduce
ultrasound image quality. With respect to drug transport,
polydispersity may prevent a precise release of active agents.
SUMMARY OF THE INVENTION
Aspects of the invention relate to microbubbles, as well as devices
and processes for producing microbubbles.
In accordance with one aspect, the invention provides a
microfluidic device for generating microbubbles. The microfluidic
device includes a substrate and a microfluidic channel embedded in
the substrate. The microfluidic channel includes a plurality of
fluid inlets, a flow focusing junction, and at least one bubble
formation outlet, the at least one bubble formation outlet
comprising a nozzle having an adjustable diameter.
In accordance with another aspect, the invention provides a method
for mass producing monodisperse microbubbles with a microfluidic
device. The method includes supplying a flow of dispersed phase
fluid into a first fluid inlet of a microfluidic channel, supplying
a flow of continuous phase fluid into a second fluid inlet of the
microfluidic channel, and adjusting a diameter of a nozzle to
obtain a plurality of monodisperse microbubbles having a specified
diameter.
In accordance with yet another aspect, the invention provides a
composition having a plurality of stable monodisperse microbubbles.
Each microbubble includes a spherical shell having a mixture of
oleosin and a surfactant, and an inner core having a gas.
In accordance with still another aspect, the invention provides a
pharmaceutical composition having a plurality of stable
monodisperse microbubbles. Each microbubble includes a spherical
shell having a mixture of oleosin and a surfactant, and an inner
core having a gas.
In accordance with still another aspect, the invention provides an
ultrasound contrast enhancing agent having a plurality of stable
monodisperse microbubbles. Each microbubble includes a spherical
shell having a mixture of oleosin and a surfactant, and an inner
core having a gas.
In accordance with a further aspect, the invention provides a
recombinant protein having the amino acid sequence selected from
SEQ ID NOS: 1-13.
In accordance with still a further aspect, the invention provides a
pharmaceutical composition having a recombinant protein having the
amino acid sequence selected from SEQ ID NOS: 1-13.
It is to be understood that both the foregoing general description
and the following detailed description are exemplary, but are not
restrictive, of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
The invention is best understood from the following detailed
description when read in connection with the accompanying drawings,
with like elements having the same reference numerals. When a
plurality of similar elements are present, a single reference
numeral may be assigned to the plurality of similar elements with a
small letter designation referring to specific elements. When
referring to the elements collectively or to a non-specific one or
more of the elements, the small letter designation may be dropped.
This emphasizes that according to common practice, the various
features of the drawings are not drawn to scale unless otherwise
indicated. On the contrary, the dimensions of the various features
may be expanded or reduced for clarity. Included in the drawings
are the following figures:
FIG. 1 is a schematic illustration of a microfluidic device
according to principles of the present invention;
FIG. 2 is a micrograph of a microfluidic device according to
principles of the present invention during the generation of
microbubbles depicting the effect of changing the size of the
nozzle on generate uniform microbubbles of different sizes;
FIG. 3 is a graph depicting microbubbles generation frequency vs.
volume of microbubbles according to principles of the present
invention;
FIG. 4 is a flow diagram of a method for mass producing
monodisperse microbubbles with a microfluidic device according to
principles of the present invention;
FIG. 5 is a schematic illustration of a microbubble according to
principles of the present invention;
FIG. 6a is an SDS-PAGE gel for 42-30G-63 according to principles of
the present invention;
FIG. 6b is a MADLI-TOF spectra confirming the molecular weight for
42-30G-63 according to principles of the present invention;
FIG. 6c is a far ultraviolet circular dichroism (UV CD) spectrum of
42-30G-63 according to principles of the present invention;
FIG. 7 is SEM images of dried microbubbles produced using a mixture
of oleosin and (PEO).sub.78-(PPO).sub.30-(PEO).sub.78 according to
principles of the present invention;
FIG. 8a is an SDS-PAGE gel for eGFP-42-30G-63 according to
principles of the present invention;
FIG. 8b is a MADLI-TOF spectra confirming the molecular weight for
eGFP-42-30G-63 according to principles of the present
invention;
FIG. 9 is micrographs of microbubbles produced using the oleosin
protein 42-30G-63 according to principles of the present
invention;
FIG. 10a is a micrograph of microbubbles produced using a mixture
of oleosin and (PEO).sub.78-(PPO).sub.30-(PEO).sub.78 present upon
tubing according to principles of the present invention;
FIG. 10b is a micrograph of microbubbles produced using a mixture
of oleosin and (PEO).sub.78-(PPO).sub.30-(PEO).sub.78 24 hours
after collection according to principles of the present
invention;
FIG. 11 is a micrograph of monodisperse microbubbles produced using
a mixture of oleosin and (PEO).sub.78-(PPO).sub.30-(PEO).sub.78 and
collected into a well in the PDMS device without the use of plastic
tubing according to principles of the present invention;
FIG. 12a is a graph depicting the size of microbubbles over time
according to principles of the present invention;
FIG. 12b is a micrograph depicting the size of microbubbles upon
collection according to principles of the present invention;
FIG. 12c is a micrograph depicting the size of microbubbles 7 days
after collection according to principles of the present
invention;
FIG. 12d is a micrograph depicting the size of microbubbles 24 days
after collection according to principles of the present
invention;
FIG. 13a is a confocal fluorescent microscopy image of bubbles
produced with oleosin according to principles of the present
invention;
FIG. 13b is a confocal fluorescent microscopy image of bubbles
produced with oleosin according to principles of the present
invention;
FIG. 13c is a confocal fluorescent microscopy image of bubbles
produced with a blend containing the eGFP mutant according to
principles of the present invention;
FIG. 13d is a confocal fluorescent microscopy image of bubbles
produced with a blend containing the eGFP mutant according to
principles of the present invention; and
FIG. 14 is ultrasound sonography images of C.sub.4F.sub.8
microbubbles generated with a solution containing 1 mg mL.sup.-1
oleosin and 10 mg mL.sup.-1-(PEO).sub.78-(PPO).sub.30-(PEO).sub.78
over time according to principles of the present invention.
FIG. 15a illustrates the generation of microbubbles by PDMS Hole
Array Method.
FIG. 15b shows the average radius of the microbubbles (Rb,avg), as
controlled by the PDMS hole sizes.
FIGS. 16a-d show microbubbles with different amounts of
Pluronic.RTM. F68 and/or Oleosin-30G:
FIG. 16a shows microbubbles with F68 at 1 mg/ml.
FIG. 16b shows microbubbles with Oleosin-30G at 1 mg/ml.
FIG. 16c shows microbubbles with Oleosin-30G at 1 mg/ml and F68 at
10 mg/ml.
FIG. 16d shows microbubbles with Oleosin-30G at 1 mg/ml and F68 at
20 mg/ml.
FIG. 16e shows changes in radius of microbubbles stabilized at
different compositions as a function of time after collection.
FIG. 17a illustrates micropipette aspiration of
oleosin-30G-stabilized microbubbles with Pluronic.RTM. F68
(Oleosin-30G at 1 mg/ml+F68 at 10 mg/ml).
FIG. 17b shows representative aspiration results, exhibiting the
typical stress-strain behavior of polymers.
FIG. 17c illustrates the Membrane Expanding Elasticity Modulus
(Ka).
FIGS. 18a-e illustrate the effect of blending concentrations of a
membrane sealing agent, Pluronic.RTM. F68:
FIG. 18a shows a real strain vs. tension for microbubbles with pure
Oleosin-30G.
FIG. 18b shows a real strain vs. tension for microbubbles with
Oleosin-30G+F68 at 10 mg/ml.
FIG. 18c shows a real strain vs. tension for microbubbles with
Oleosin-30G+F68 at 20 mg/ml.
FIG. 18d illustrates the increase in slope as F68 concentration
increases in solution.
FIG. 18e illustrates the amount of F68 vs. modulus.
FIGS. 19a and 19b illustrate the effects on mechanical properties
of Oleosin-30G microbubbles by adding different kinds of
Pluronic.RTM. surfactants:
FIG. 19a illustrates a real strain vs. tension with different kinds
of Pluronic.RTM. surfactants.
FIG. 19b illustrates variations in modulus with different kinds of
Pluronic.RTM. surfactants.
FIG. 20 illustrates physical properties of Oleosin 30G (MW=15,206
g/mol) and Pluronic.RTM. surfactants (F68 MW=8,400, F77 MW=6,600,
P105 MW=6,500, L64 MW=2,900).
DETAILED DESCRIPTION OF THE INVENTION
Aspects of the invention are directed to stable monodisperse
microbubbles, methods for producing stable monodisperse
microbubbles, stable monodisperse microbubbles produced by the
inventive methods, and microfluidic devices for producing stable
monodisperse microbubbles.
The inventors have recognized that it would be useful to provide
stable and monodisperse protein-shelled microbubbles using a
microfluidic flow focusing device. The inventors have also
recognized that the use of a microfluidic device having an outlet
nozzle with an adjustable diameter enables the production of highly
monodisperse microbubbles, even at diameters below 10 .mu.m. In
particular, the inventors have found that the use of an
fluid-actuated membrane valve enables precise control over the size
of microbubbles while producing highly monodisperse
microbubbles.
The inventors have also recognized that the use of particular
recombinant proteins, such as oleosin, in the microbubble shell
results in highly stable microbubbles. The inventors have further
recognized that this use of, e.g., oleosin provides versatility in
controlling the mechanical properties of the microbubble shell and
adding specific ligands for targeted drug delivery applications
through recombinant biotechnology.
As used herein, "monodisperse" means that the polydispersity index
("PDI") for a given collection of microbubbles is less than 5%. PDI
is mathematically defined as PDI=s/n, wherein n denotes the average
microbubble radius and s is the standard deviation of the bubble
radii.
As used herein, "functionalized," "functionalization," "or
"modified," when used to refer to oleosin, means that the oleosin
has been altered using recombinant protein techniques to have
different functionality and properties. For example, oleosin may be
modified to include specific motifs or targeting ligands such as
protease sites, adhesion peptides or affibodies. In one example,
described in more detail below, green fluorescent protein (eGFP) is
fused to the N-terminus of an oleosin mutant. One of ordinary skill
in the art will understand that functionalization, however, may
occur at any point of the oleosin or oleosin mutant. Oleosin may be
functionalized using one or more of the following recombinant
protein techniques: recombinant biotechnology, enzymatic linking,
or direct covalent bonds. One of ordinary skill in the art will
understand that other techniques may be used to achieve these
alterations.
As used herein, "microfluidic channel" refers collectively to all
channels in fluid communication with the continuous phase fluid
inlet and the dispersed phase fluid inlet.
As used herein, "oleosin" refers to either a homogenous population
of the same species of oleosin protein, a heterogeneous population
of different species of oleosin proteins, or mutants thereof. For
example, a substantial number of oleosin protein sequences, and
associated nucleotide sequences encoding, are known from a large
number of different plant species. Examples include, but are not
limited to, oleosins from Arabidposis, canola, corn, rice, peanut,
castor, soybean, flax, grape, cabbage, cotton, sunflower, sorghum
and barley. While the present disclosure uses a sunflower seed
oleosin gene for the purposes of illustrating certain principles of
the invention, one of ordinary skill in the art will understand
that the term "oleosin" is used broadly herein to refer not only to
the explicitly described oleosin species and mutants, but to other
oleosin species and oleosin mutants.
FIG. 1 shows a schematic perspective view of an exemplary
embodiment of a microfluidic device 100 for generating monodisperse
microbubbles according to aspects of the present invention.
Microfluidic device 100 may be formed on a substrate. Exemplary
substrates materials include polysiloxanes or carbon-based polymers
including, but not limited to polydimethylsiloxane ("PDMS"), a
polyacrlyamide, a polyacrylate, a polymethacrylate or a mixtures
thereof.
Microfluidic device 100 includes at least two fluid inlets embedded
in the substrate, including at least one continuous fluid inlet 110
and at least one dispersed fluid inlet 120. Continuous phase fluid
inlet 110 and dispersed fluid inlet 120 are in fluid communication
and may join one another at flow focusing junction 125. Bubble
formation outlet 137 is similarly in fluid communication with
continuous phase fluid inlet 110, dispersed phase fluid inlet 120,
and flow focusing junction 125. Because each are in continuous
fluid communication with each other, bubble formation outlet 137,
continuous phase fluid inlet 110, dispersed phase fluid inlet 120,
and flow focusing junction 125 are collectively referred to as a
"microfluidic channel."
The microfluidic channel is, preferably, entirely enclosed within
the substrate. Additionally, the microfluidic channel may, as
depicted have different cross-sectional geometries at different
locations. For example, continuous fluid supply channels 115 may
have a rectangular cross-sectional geometry, but other geometries
known to one of ordinary skill in the art will also be understood
to be within the scope of the present invention. Other
cross-sectional geometries include circular, octagonal, and other
polygonal designs.
Each of bubble formation outlet 137, continuous phase fluid inlet
110, dispersed phase fluid inlet 120, and flow focusing junction
125 have a hydraulic diameter that is preferably smaller than 100
.mu.m.
Continuous phase fluid inlet 110 supplies microfluidic device 100
with a controlled flow of a continuous phase fluid such as a
liquid. In one embodiment, continuous fluid inlet 110 branches into
two continuous fluid supply channels 115 which converge again at
flow focusing junction 125.
Dispersed phase fluid inlet 120 supplies microfluidic device 100
with a controlled flow of a dispersed phase fluid such as a
gas.
In an exemplary embodiment, continuous phase fluid inlet 110 and
dispersed phase fluid inlet 120 discharge into flow focusing
junction 125. Upon mixing of these inlets at flow focusing junction
125, microbubbles 139 are generated at nozzle 130 of bubble
formation outlet 137. Microbubbles 139 then flow towards collection
unit 150 for subsequent recovery.
Nozzle 130 preferably has an adjustable diameter. In particular,
according to an aspect of this embodiment of the present invention,
a user of microfluidic device 100 can dynamically tune the channel
diameter of bubble formation outlet 137 at nozzle 130. In one
embodiment, a fluid-actuated membrane valve 135 is used to
constrict/expand nozzle 130 of bubble formation outlet 137 to
obtain a desired diameter. The diameter of nozzle 130 may be
adjusted through the application of pressure to valve 135. Pressure
may be supplied to valve 135 via valve actuation inlet 140. In the
depicted embodiment, fluid-actuated membrane valve 135 encircles
nozzle 130, such that the application of pressure causes the valve
135 to inflate, thereby constricting the diameter of nozzle 130.
Preferably, fluid-actuated membrane valve 135 is not in fluid
communication with the microfluidic channel.
The use of fluid-actuated membrane valve 135 enables the control
over the size of monodisperse bubbles. Both liquids and gases are
suitable fluids to actuate membrane valve 135. In one embodiment,
fluid-actuated membrane valve is an air-actuated membrane valve.
This flexible design permits a user of microfluidic device 100 to
tune the size of the microbubbles 139 without changing the
continuous phase or dispersed phase flow rates, by only changing
the size of nozzle 130 through the application of pressure to valve
135.
In one embodiment, the microfluidic device may be configured to
have more than one bubble formation outlet, with each bubble
formation outlet having a nozzle with an adjustable diameter.
Advantageously, from the perspective of manufacture, microfluidic
device 100 may be constructed such that the microfluidic channel
and valve 135 exist in the same plane. Doing so permits fabrication
of microfluidic device 100 in a single layer mold. The use of a
single layer membrane valve also overcomes the low resolution that
is typically achieved by using polymeric photomasks (which are
typically limited to ranges of microbubbles above 10 .mu.m).
However, the present invention is not limited to a planar flow
focusing geometry, and one of ordinary skill in the art will
understand that other geometries fall within the scope of the
invention disclosed herein. For example other potential geometries
include glass capillary devices, etched glass devices or 3-D PDMS
devices.
As described above, microfluidic device 100 may be used to produce
monodisperse microbubbles having tunable radii and a narrow size
distribution. That is, microfluidic device 100 may be used to
produce monodisperse microbubbles with radii ranging from 0.5 to 10
.mu.m. By using a single microfluidic device according to aspects
of the present invention, microbubbles having a broad range of
radii may be generated, unlike most presently available
flow-focusing microfluidic devices. In particular, as shown by FIG.
2, increasing the pressure that is applied to valve 145 decreases
the orifice diameter of the nozzle 133 and, in turn, decreases the
size of microbubbles. According to FIG. 2, the diameter of the
microbubbles, d.sub.b, decreases linearly with the width of the
nozzle w.sub.n. Thus, the size of microbubbles 139 may be precisely
tuned by dynamically changing the dimension of nozzle 133 using
valve 145.
Microbubble generation frequency (f=the number of microbubbles
generated per second) is shown, in FIG. 3, to be inversely
proportional to the volume of microbubbles, e.g.,
f.about.d.sub.b.sup.-3. This trend indicates that the gas flow rate
remains generally constant under varying nozzle size.
Turning to FIG. 4, a flow diagram depicting selected steps of a
process 400 for producing stable monodisperse microbubbles using a
microfluidic device according to aspects of the invention is shown.
It should be noted that, with respect to the methods described
herein, it will be understood from the description herein that one
or more steps may be omitted and/or performed out of the described
sequence of the method (including simultaneously) while still
achieving the desired result.
In step 410, a flow of a dispersed phase fluid is supplied into a
first fluid inlet (e.g., dispersed phase fluid inlet 120; FIG. 1)
of a microfluidic channel. In one embodiment, the dispersed phase
fluid is a gas. The gas may be an inert gas. In particular, the gas
may be one or more of nitrogen, carbon dioxide, helium, neon,
xenon, argon, air, oxygen, sulfur hexafluoride, or heavy per
fluorocarbon gases such as octafluorocyclobutane. For medical
applications, gases having less solubility in water (e.g.,
nitrogen, air, sulfur hexafluoride and heavy perfluorocarbon gases)
are preferred as this causes bubble dissolution to occur at a
slower rate.
In step 420, a flow of a continuous phase fluid is supplied into a
second fluid inlet (e.g., continuous phase fluid inlet 110; FIG. 1)
of the microfluidic channel. In one embodiment, the continuous
phase fluid is a liquid or liquid mixture. For example, and as
described more fully below, the continuous phase fluid may be a
mixture of a recombinant protein, such as oleosin, and a
surfactant, such as a triblock copolymer.
In step 430, the diameter of a nozzle through which the mixture of
dispersed phase and continuous phase fluids passes is adjusted to
obtain a plurality of monodisperse microbubbles having a specified
diameter. As described above, continuous phase fluid inlet 110 and
dispersed phase fluid inlet 120 may discharge into flow focusing
junction 125 that leads to nozzle 130 of bubble formation outlet
137. In the embodiment described above, an air-actuated membrane
valve may be used to constrict/expand the nozzle to obtain a
desired diameter. The fluid actuated membrane valve may encircle
the nozzle, such that the application of pressure causes the valve
to inflate, thereby constricting the diameter of nozzle.
In accordance with other aspects, a plurality of microbubbles is
provided. The plurality of microbubbles may be obtained from the
inventive methods described herein.
In accordance with other aspects, a composition including a
plurality of stable monodisperse microbubbles is provided. Turning
to FIG. 5, a microbubble 500 according to aspects of the present
invention is shown. The microbubbles may be stabilized by
incorporating an amphiphilic protein, oleosin, into spherical
microbubble shell 510. Microbubbles incorporating oleosin are also
echogenic and thus have value with respect to theranostic
applications. Microbubbles composed of gaseous cores covered with
stabilizing agents, such as oleosin, can drastically enhance the
ultrasound signal because of their large compressibility, which
leads to enhanced scattering of ultrasound.
Oleosins are structural proteins which are found in, and stabilize,
vascular plant oil bodies. The protein has a natural amphiphilic
structure (i.e., it includes both hydrophilic and hydrophobic
groups). Oleosin proteins are composed of three distinctive
domains: a central hydrophobic portion between N terminal and
C-terminal amphiphilic arms. The central hydrophobic portion
contains a proline knot which forces the protein into a hairpin
structure. The elimination of a large portion of the hydrophobic
domain and removal of the secondary structure in the protein
backbone has been shown to yield a soluble oleosin mutant that
naturally self-assembles into miscelles. This soluble oleosin
mutant is named 42-30-63, which refers to the number of amino acids
in each domain: the N-terminal arm, the central hydrophobic core,
and the C-terminal hydrophilic arm, respectively. 42-30-63 may be
produced by truncating the wildtype molecule without changes in the
sequence of amino acids.
In one embodiment, a further modification of the 42-30-63 oleosin
mutant is used in microbubble shell 510. In particular, the
42-30-63 oleosin mutant was further modified by inserting five
glycines into the hydrophobic core, as shown by the amino acid
sequence set forth in SEQ ID NO: 1, creating a mutant referred to
as 42-30G-63. The addition of the five glycines desirably increases
the protein expression, stability, and solubility while abolishing
secondary structure, as shown by circular dichroism depicted in
FIG. 6c. The protein was expressed in the Escherichia coli strain
BL21 (DE3) with isopropyl .beta.-D-1-thiogalactopyranoside (IPTG)
induction.
In certain embodiments, the oleosin is functionalized. For example,
as described above, oleosin may be modified to include specific
targeting ligands such as binding motifs or affibodies.
Microbubbles having these targeting ligands may be used to deliver
an active pharmaceutical ingredient in higher concentrations to
particular parts of a patient's body. For example, by
functionalizing oleosin with specific targeting ligands via
recombinant protein techniques, it will be possible to enable
localized antivascular ultrasound therapy. Recombinant protein
techniques may also be used to alter the molecular structure of
oleosin (e.g., control the structure of the hydrophobic domain),
thereby generating microbubble shells having different rheological
properties. For example, alpha-helical domains, hydrogen bond
networks, or crosslinking sites can be mutated into the hydrophobic
core of oleosin increasing lateral interactions in the membrane
potentially modifying elastic properties of the bubble shell.
Oleosin also provides the inventive microbubbles with versatility
in that it enables the functionalization of microbubbles through
recombinant biotechnology. By contrast, most microbubbles that are
currently being developed use stabilizing agents such as
phospholipids, proteins and polymers that undesirably cannot be
easily modified to have, e.g., targeting ligands on the microbubble
surface or to enable the modulation of the rheological properties
of the stabilizing shells.
Spherical shell 510 may include a mixture of oleosin and one or
more additional components, such as a surfactant. In one
embodiment, oleosin is combined with a surfactant such as a
triblock copolymer. For example, oleosin may be combined with a
triblock copolymer of poly(ethylene glycol)-b-poly(propylene
glycol)-b-poly(ethylene glycol)
((PEO).sub.n-(PPO).sub.m-(PEO).sub.n where n and m denote the
number of ethylene oxide and propylene oxide repeat units,
respectively; these polymers are also known as Pluronic and
Polxamer). Other suitable surfactants include phospholipids,
diblock copolymers, non-ionic surfactants, ionic surfactants. The
combination of oleosin and surfactants has been found to have a
particularly favorable effect on microbubble stability and
generation.
Microbubble 500 also includes an inner core 520 filled with a
dispersed phase, such as a gas. Inert gases are generally suitable
for use in inner core 520. Exemplary gases include N.sub.2 or
C.sub.4F.sub.8 and CO.sub.2. FIG. 5 depicts a mixture of N.sub.2
and C.sub.4F.sub.8.
The composition of microbubbles may be dried through conventional
methods (e.g., freeze drying), stored, and then rehydrated for
later use. FIG. 7 depicts SEM images of dried microbubbles produced
using a mixture of oleosin and
(PEO).sub.78-(PPO).sub.30-(PEO).sub.78. Nearly full and rapid
rehydration of the inventive microbubbles after storage in dry
state for several months has been achieved.
In accordance with other aspects, a pharmaceutical composition
having a plurality of stable monodisperse microbubbles is provided.
Each microbubble includes a spherical shell having a mixture of
oleosin and a surfactant, and an inner core filled with gas.
In accordance with other aspects, an ultrasound contrast enhancing
agent having a plurality of stable monodisperse microbubbles is
provided. Each microbubble includes a spherical shell having a
mixture of oleosin and a surfactant, and an inner core filled with
gas.
In accordance with other aspects, a recombinant protein having an
amino acid sequence selected from the group consisting of SEQ ID
NOS: 1-13 is provided.
According to particular embodiments, the oleosin described herein
comprises, consists essentially of, or consists of an amino acid
sequence selected from the group consisting of SEQ ID NOS:
1-13.
According to particular embodiments, one or more of the amino acids
in SEQ ID NOS: 1-13 may be replaced with one or more different
amino acids (e.g., between 1 and 10 amino acids).
According to particular embodiments, the oleosin may have at least
80% homology, or at least 85% homology, or at least 90% homology,
or at least 95% homology, or at least 97% homology to any of the
sequences selected from the group consisting of SEQ ID NOS:
1-13.
SEQ ID NO: 3 is a wild type oleosin sequence, with an N-terminal
hydrophilic domain of 42 amino acids (starting with M), a central
block of 87 amino acids (underlined), and a C-terminal block of 63
amino acids, including six H residues added for purification (his
tag).
SEQ ID NOS: 4-9 are various truncations/modifications of SEQ ID NO:
3, wherein X may be any naturally-occurring or artificial amino
acid and any number of the X amino acids may be absent (i.e., any
one, more than one, or all of the X amino acids may be present or
absent). The "central block" of amino acids is underlined in each
of SEQ ID NOS: 3-10 shown below.
In SEQ ID. NO: 4, the N-terminal sequence is kept the same and the
C-terminal sequence is kept the same, and the number of amino acids
in the central (underlined) sequence can be changed, preferably
from 87 down to 29 amino acids, or any number in between. X at
positions 43 to 129 may be any naturally-occurring or artificial
amino acid and up to 87 of them may be absent.
In SEQ ID. NO: 5, the N-terminal sequence can be changed,
preferably between 1 and 42 amino acids, the C-terminal sequence is
kept the same, and the central (underlined) sequence is kept the
same. X at positions 1 to 42 may be any naturally-occurring or
artificial amino acid and up to 42 of them may be absent.
In SEQ ID. NO: 6, the N-terminal sequence is kept the same, the
central (underlined) sequence is kept the same, and the C-terminal
sequence can be changed, preferably between 1 and 63 amino acids. X
at positions 130 to 192 may be any naturally-occurring or
artificial amino acid and up to 63 of them may be absent.
In SEQ ID. NO: 7, only the C-terminal sequence is kept the same. X
at positions 1 to 129 may be any naturally-occurring or artificial
amino acid and up to 129 of them may be absent.
In SEQ ID. NO: 8, only the N-terminal sequence is kept the same. X
at positions 43 to 192 may be any naturally-occurring or artificial
amino acid and up to 150 of them may be absent.
In SEQ ID. NO: 9, only the central block is kept the same. X at
positions 1 to 42 may be any naturally-occurring or artificial
amino acid and up to 42 of them may be absent; and X at positions
130 to 192 may be any naturally-occurring or artificial amino acid
and up to 63 of them may be absent.
In SEQ ID. NO: 10, the central block (underlined) of SEQ ID. NO: 3
is truncated to 29 amino acids.
In SEQ ID. NO: 11, N- and C-termini were modified with individual
amino acids to make the sequences more negatively charged, called
Oleosin(-).
In SEQ ID. NO: 12, N- and C-termini were modified with individual
amino acids to make the sequences more positively charged, called
Oleosin(+).
SEQ ID. NO: 13 is another oleosin sequence wherein X may be any
naturally-occurring or artificial amino acid and any number of the
X amino acids may be absent (i.e., X at positions 133 to 138 may be
any naturally-occurring or artificial amino acid and up to 6 of
them may be absent). According to one embodiment, XXXXXX is RGDS
(for binding a receptor).
According to particular embodiments, an affibody may be appended to
either end of any of the oleosin sequences (SEQ ID NOS: 1-13)
described herein (e.g., an affibody of length 5 to 80 amino acids).
According to particular embodiments, the affibody is the Her-2
Affibody having SEQ ID NO: 14.
According to particular embodiments, any of the oleosin sequences
described herein may be absent the starting methionine, due to
methionine cleavage upon expression.
In accordance with other aspects, a pharmaceutical composition
having a recombinant protein having the amino acid sequence
selected from the group consisting of SEQ ID NOS: 1-13 is
provided.
Oleosin Sequences
TABLE-US-00001 SEQ ID NO: 1
GSATTTYDRHHVTTTQPQYRHDQHTGDRLTHPQRQQQGPSTGKLALGATP
LFGVIGFSPVIVPAMGIAIGLAGVTGFQRDYVKGKLQDVGEYTGQKTKDL
GQKIQHTAHEMGDQGQGQGQGGGKEGRKEGGKLEHHHHHH SEQ ID NO: 2
VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTT
GKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFF
KDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNV
YIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHY
LSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGSATTTYDRHHV
TTTQPQYRHDQHTGDRLTHPQRQQQGPSTGKLALGATPLFGVIGFSPVIV
PAMGIAIGLAGVTGFQRDYVKGKLQDVGEYTGQKTKDLGQKIQHTAHEMG
DQGQGQGQGGGKEGRKEGGKLEHHHHHH SEQ ID NO: 3
MATTTYDRHHVTTTQPQYRHDQHTGDRLTHPQRQQQGPSTGKIMVIMALL
PITGILFGLAGITLVGTVIGLALATPLFVIFSPVIVPAMIAIGLAVTGFL
TSGTFGLTGLSSLSYLFNMVRRSTMSVPVQRDYVKGKLQDVGEYTGQKTK
DLGQKIQHTAHEMGDQGQGQGQGGGKEGRKEGGKLEHHHHHH SEQ ID NO: 4
MATTTYDRHHVTTTQPQYRHDQHTGDRLTHPQRQQQGPSTGKXXXXXXXX
XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX
XXXXXXXXXXXXXXXXXXXXXXXXXXXXXQRDYVKGKLQDVGEYTGQKTK
DLGQKIQHTAHEMGDQGQGQGQGGGKEGRKEGGKLEHHHHHH SEQ ID NO: 5
XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXIMVIMAL
DLPITGILFGLAGITLVGTVIGLALATPLFVIFSPVIVPAMIAIGLAVTG
FLTSGTFGLTGLSSLSYLFNMVRRSTMSVPVQRDYVKGKLQDVGEYTGQK
TKDLGQKIQHTAHEMGDQGQGQGQGGGKEGRKEGGKLEHHHHHH SEQ ID NO: 6
MATTTYDRHHVTTTQPQYRHDQHTGDRLTHPQRQQQGPSTGKIMVIMALL
PITGILFGLAGITLVGTVIGLALATPLFVIFSPVIVPAMIAIGLAVTGFL
TSGTFGLTGLSSLSYLFNMVRRSTMSVPVXXXXXXXXXXXXXXXXXXXXX
XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX SEQ ID NO: 7
XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX
XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX
XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXQRDYVKGKLQDVGEYTGQK
TKDLGQKIQHTAHEMGDQGQGQGQGGGKEGRKEGGKLEHHHHHH SEQ ID NO: 8
MATTTYDRHHVTTTQPQYRHDQHTGDRLTHPQRQQQGPSTGKXXXXXXXX
XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX
XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX
XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX SEQ ID NO: 9
XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXIMVIMAL
LPITGILFGLAGITLVGTVIGLALATPLFVIFSPVIVPAMIAIGLAVTGF
LTSGTFGLTGLSSLSYLFNMVRRSTMSVPVXXXXXXXXXXXXXXXXXXXX
XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX SEQ ID NO: 10
MATTTYDRHHVTTTQPQYRHDQHTGDRLTHPQRQQQGPSTGKLALATPLF
VIFSPVIVPAMIAIGLAVTGFQRDYVKGKLQDVGEYTGQKTKDLGQKIQH
TAHEMGDQGQGQGQGGGKEGRKEGGKLEHHHHHH SEQ ID NO: 11
GSEATTTNDQHHVTTTQPQDQHDQHTGDQLTHPQDQQQGPSTGELALGAT
PLFGVIGFSPVIVPAMGIAIGLAGVTGFQWQDNVNGELQDVGEQTGQNTN
DLGQQIQHTAHEMGDQGQGQGQGGGNEGQNEGGNHHHHHHDD SEQ ID NO: 12
GSATTTKNRHHVTTTQPQKRHNQHTGNRLTHPQRQQQGPSTGKLALGATP
LFGVIGFSPVIVPAMGIAIGLAGVTGFQWNKVKGKLQNVGQKTGQKTKNL
GQKIQHTAHQMGNQGQGQGQGGGKQGRKQGGKLEHHHHHH SEP ID NO: 13
GSTTTYDRHHVTTTQPQYRHDQHTGDRLTHPQRQQQGPSTGKLALATPLF
VIFSPVIVPAMIAIGLAVTGFQRDYVKGKLQDVGEYTGQKTKDLGQKIQH
TAHEMGDQGQGQGQGGGKEGRKEGGKHHHHHHXXXXXX (Her-2 Affibody) SEQ ID NO:
14 VDNKFNKEMRNAYWEIALLPNLNNQQKRAFIRSLYDDPSQSANLLAEAKK
LNDAQAPKLE
EXAMPLES
The following examples are included to demonstrate the overall
nature of the present invention. The examples further illustrate
the improved results obtained by generating stable monodisperse
microbubbles and by employing the microfluidic device and related
processes according to principles of the present invention.
Example 1
Manufacture of a Microfluidic Device
Microfluidic flow focusing devices with expanding nozzle design
according to FIG. 1 were fabricated using single layer soft
lithography in PDMS. Negative photoresist SU-8 2010 (Microchem,
Newton, Mass.), thinned to a 3:1 ratio with SU-8 developer, was
spin coated onto a clean silicon wafers to a thickness of 5 .mu.m
and patterned to UV light through a transparency photomask (CAD/Art
Service, Bandon, Oreg.) using a Karl Suss MA4 Mask Aligner (SUSS
MicroTec Inc., Sunnyvale, Calif.). To incorporate an fluid-actuated
valve, single-layer membrane valves, described by A. R. Abate et
al., Appl. Phys. Lett. 2009, 94, 023503, were used. The single
layer membrane exists in the same plane as the microfluidic
channel, which permitted fabrication of the entire microfluidic
device in a single layer mold. Sylgard 184 poly(dimethylsiloxane)
(Dow Corning, Midland, Mich.) was mixed with crosslinker (ratio
12:1), degassed thoroughly and poured onto the photoresist pattern,
and cured for 1 hr at 65.degree. C. to make the membrane highly
compliant. The PDMS replica were peeled off the wafer and bonded to
a PDMS membrane fabricated by spin coating PDMS on a glass slide
after oxygen-plasma activation of both surfaces. Having a
microchannel fully-enclosed in PDMS allows for more efficient use
of the valve-membrane.
Example 2
Gene Creation and Protein Expression
The sunflower seed oleosin gene was provided as a gift from Dr.
Beaudoin at Rothamsted Research, Hampshire, England. Multiple
rounds of PCR were used to create the oleosin gene 42-30G-63 and
eGFP 42-30G-63. Multiple rounds of PCR were used to create the
oleosin gene 42-30G-63 and eGFP-42-30G-63. The following PCR
primers were used to create the three domains, which were combined
in a single PCR step: N-terminal hydrophilic S
5'-AAGGAGATAGGATCCACCACAACCTACGACC-3' (SEQ ID NO: 15), N-terminal
hydrophilic AS 5'-GCACCGAGAGCGAGCTTGCCGGTFGAGG-3' (SEQ ID NO: 16),
hydrophobic S 5'-CCTCAACCGGCAAGCTCGCTCTCGGTGC-3' (SEQ ID NO: 17),
hydrophobic AS 5-CCTTCACATAATCCCTCTGAAACCCGGTAACACC-3' (SEQ ID NO:
18), C-terminal hydrophilic S
5'-GGTGTTACCGGGTTTCAGAGGGATTATGTGAAGG-3' (SEQ ID NO: 19),
C-terminal hydrophilic AS 5'-TATATGAATCTCGAGTTTCCCCCCTTCHTTTTCG-3'
(SEQ ID NO: 20). The PCRs to create the hydrophilic portions were
run with the soluble oleosin gene as the template and the
hydrophobic domain PRC was run with the following oligo as the
template:
5'-CTCGCTCTCGGTGCGACTCCGCTGTTTGGTGTTATAGGITTCAGCCCTGTTATTGTTCCAGCGAT
GGGTATAGCGATTGGGCTTGCGGGTGTTACCGGGTTTCAG-3' (SEQ ID NO: 21). PCR
was used to create the eGFP mutants using the following primers:
eGFP S 5'-ATCGGTATACATATGGTGAGCAAGGGCGAGG-3' (SEQ ID NO: 22) and
eGFP AS 5'-ATCTAAAATGGATCCCTTGTACAGCTCG-3' (SEQ ID NO: 23) with
pBamUK-eGFP as a template. The genes were inserted into the
expression vector pBamUK, a pET series derivative constructed by
the Duyne Laboratory (School of Medicine, University of
Pennsylvania).
Mutants were confirmed through DNA sequencing prior to protein
expression. pBamUK adds a 6-Histidine tag to the C-terminus of the
protein for IMAC purification. Protein was expressed in the E. Coli
strain BL21 DE3 (Stratagene) controlled by the lac promoter.
Cultures were grown at 37.degree. C. in Luria Bertani (LB) with
kanamycin (50 .mu.g ml.sup.-1) until OD.sub.600.apprxeq.0.7-0.9.
Protein expression is induced with isopropyl
.beta.-D-1-thiogalactopyranoside (IPTG) to a final concentration of
1.0 mM. Cells were harvested by centrifugation and cell pellets are
frozen at -20.degree. C. prior to purification.
Example 3
Protein Purification and Characterization
B-PER protein extraction agent (Fisher Scientific, Waltham, Mass.)
was used for protein purification. Pellets were resuspended in
B-PER (30 ml B-PER per liter of culture) and DNAse was added to a
final concentration of 1 .mu.g/ml. The resuspended pellets were
centrifuged at 15,000 g for 15 minutes. The 42-30G-63 supernatant
was discarded and the eGFP-42-30G-63 supernatant was applied to an
equilibrated column and allowed to bind for >1 hour. The
remaining inclusion body pellet of 42-30G-63 was suspended in
denaturing buffer (8M urea, 50 mM phosphate buffer, 300 mM NaCl).
The solution was centrifuged at 15,000 g for 15 minutes and the
supernatant was added to an equilibrated Ni-NTA column (Hispur
Ni-NTA resin, Thermo Scientific). The denatured 42-30G-63 was
allowed to bind to the column for >1 hours and washed three
times with denaturing wash buffer (denaturing buffer with 20 mM
imidazole). 42-30G-63 refolding was accomplished by diluting the
column 50 times with refolding buffer (50 mM phosphate buffer, 300
mM NaCl, 5% by volume glycerol, 4.degree. C.) and rocked at
4.degree. C. for >1 hr. Both mutants was washed extensively with
wash buffer (50 mM phosphate buffer, 300 mM NaCl, 20 mM imadzole)
and eluted in fractions with elution buffer (50 mM phosphate
buffer, 300 mM NaCl, 300 mM imidazole).
The concentration of purified protein was measured with a Nano-Drop
1000 (Thermo Scientific, Philadelphia, Pa.). Buffer exchange was
completed with dialysis. All analysis was completed in PBS unless
otherwise noted. To establish the purity of the proteins, SDS/PAGE
gels were run on NuPAGE Novex 4-12% Bis-Tris mini gels (Invitrogen,
Waltham, Mass.) in MES buffer. The gel was stained with SimplyBlue
SafeStain (Invitrogen, Waltham, Mass.) following electrophoresis.
The gel was destained overnight in water and imaged with a Kodak
Gel Logic 100 Imaging System. Protein molecular weight was
confirmed with MALDI-TOF. Sample spots were created with 0.5 .mu.l
protein in 1.times. PBS and 0.5 .mu.l saturated sinapinic acid
solution (50/50 acetonitrile/water+0.1% TFE). Spectra were
collected on an Ultraflextreme MALDI-TOF (Bruker, Billerica,
Mass.). FIG. 8 depicts the spectra for eGFP-42-30G-63. To measure
the protein secondary structure, far-UV CD spectra were collected
at 25.degree. C. on an AVIV 410 spectrometer (AVIV Biomedical Inc.,
Lakewood Township, N.J.) using a 1 mm quartz cell. Protein
concentration is 15 .mu.M in 50 mM phosphate, 140 mM NaF. NaF is
used to replace NaCl due to the strong absorbance of the Cl.sup.-
ion.
Example 4
Initial Testing of Microfluidic Device
For the initial testing of the microfluidic device to control the
size of microbubbles, nitrogen gas and a common surfactant, sodium
dodecyl sulfate (SDS, Sigma-Aldrich, St Luis, Mo., USA), was used
at a concentration of 20 mg mL.sup.-1 in the aqueous phase to
stabilize microbubbles. Monodisperse microbubbles were produced
with radii ranging from approximately 2 to 10 .mu.m for several
hours without changes in the bubble size. Although SDS enables the
investigation of microfluidic device performance, microbubbles
formed using SDS are not stable upon collection.
To produce stable microbubbles with high monodispersity, size
tunability and structural modularity, a structurally modified
recombinant oleosin was used as the microbubble shell material. The
42-30G-63 protein expressed in the Escherichia coli strain BL21
(DE3) with isopropyl .beta.-D-1-thiogalactopyranoside (IPTG)
induction. Protein was purified using immobilized metal affinity
chromatography through a 6-histidine tag on the C-terminus of the
protein leading to highly purified products as shown in FIGS. 8a
and 8b. Protein molecular weight is confirmed with
SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and
matrix-assisted laser desorption/ionization-time of flight
(MALDI-TOF) mass spectroscopy.
When microbubbles are produced using oleosin at concentrations
between 1-2 mg mL.sup.-1, bubbles with radii above 10 .mu.m are
stable. During the generation of microbubbles with radii smaller
than 10 .mu.m, bubbles are observed to undergo coalescence within
and outside of the microfluidic device as shown in FIG. 9. In
addition, the relatively high surface tension between the liquid
and the gas phases makes the generation of such microbubbles
challenging, often resulting instability of microbubbles in the
microfluidic device.
Poly(ethylene glycol)-b-poly(propylene glycol)-b-poly(ethylene
glycol) triblock copolymers ((PEO).sub.n-(PPO).sub.m-(PEO).sub.n
were added to the oleosin solution to test whether the production
of microbubbles can be facilitated. Two different types of
(PEO).sub.n-(PPO).sub.m-(PEO).sub.n triblock copolymers:
(PEO).sub.100-(PPO).sub.65-(PEO).sub.100 and
(PEO).sub.78-(PPO).sub.30-(PEO).sub.78 were tested. When a mixture
containing 1-2 mg mL.sup.-1 oleosin and 5-20 mg mL.sup.-1
(PEO).sub.100-(PPO).sub.65-(PEO).sub.100 (average molecular weight
12600) was used, monodisperse microbubbles were consistently
generated at the nozzle; however, these microbubbles underwent
significant coalescence upon collection. In contrast, when
(PEO).sub.78-(PPO).sub.30-(PEO).sub.78 (average molecular weight
8400) was added to oleosin solutions, microbubbles were generated
at the nozzle and very limited coalescence was observed upon
collection. A suitable concentration for stable microbubble
formation includes an aqueous phase containing 1 mg mL.sup.-1 of
oleosin and 10 mg mL.sup.-1 of
(PEO).sub.78-(PPO).sub.30-(PEO).sub.78. In the samples that are
collected through polyethylene tubing, a small number of fairly
large bubbles (>20 .mu.m in diameter) were observed. Although
the physical mechanism behind the appearance of these large bubbles
is not known, their number fraction is extremely small, typically
less than 1%. Interestingly, these large bubbles disappear
completely approximately 24 h after collection, leaving behind a
collection of highly monodisperse microbubbles as shown in FIGS.
10a and b.
Since no major coalescence was observed between microbubbles
occurring within the PDMS microfluidic device, while not intending
to be bound to a particular theory, it is believed that these large
bubbles likely form during transfer of the microbubbles from nozzle
to a container via polyethylene tubing. Possibly, abrupt changes in
dimensions and relative shear stress experienced by microbubbles
between the PDMS device and the collection tube, as well as the
lower speed at which the microbubbles travel in the polyethyelene
tube before being released in a petri dish may lead to collision
between bubbles and eventual coalescence. Another possibility is
that these large bubbles have slightly different surface
composition since they are observed to undergo dissolution when
they are stored for an extended period, whereas the monodisperse
bubbles that were originally generated at the nozzle do not
dissolve completely over a long period of time. Highly monodisperse
microbubbles are able to be collected, however, without any large
bubbles if the produced bubbles are collected straight into a well
that is positioned in the same plane as the microfluidic channel as
shown in FIG. 11. These results show that even small perturbations
can lead to disruption of microbubbles that are generated using
microfluidic devices and extra care must be taken in collecting
microbubbles for clinical applications since large bubbles in blood
vessels can lead to serious problems such as embolism.
Microbubbles generated using the mixture of oleosin and
(PEO).sub.78-(PPO).sub.30-(PEO).sub.78 (molar ratio of
oleosin:triblock copolymer=1:18) were stable once collected. When
microbubbles were collected and stored in water (microbubbles
reside at the air-water interface due to their bouyancy),
microbubble radius decreases by about 13% during the first few days
and eventually ceased to shrink further. These microbubbles remain
stable at least for 4 weeks as depicted by FIGS. 12a-d. SEM images
depict the size of the microbubbles at collection (12(b)), over 7
days (12(c)), and after 24 hours (12(d)). The stability of these
microbubbles does not depend on whether N.sub.2 or C.sub.4F.sub.8
is used as the gas phase. In contrast, microbubbles generated
solely with (PEO).sub.78-(PPO).sub.30-(PEO).sub.78 do not exhibit
such excellent stability. These results indicate that oleosin plays
a role in stabilizing the shell of microbubbles, which likely
consists of a mixture of oleosin and
(PEO).sub.78-(PPO).sub.30-(PEO).sub.78, to prevent complete
dissolution or coalescence of microbubbles upon their collection.
Similar examples, in which shells suppresses the dissolution of
microbubbles, have been observed in microbubbles that have been
stabilized with other types of proteins, nanoparticles or synthetic
polymers.
As discussed above, one of the unique aspects of oleosin is that
the molecular structure and thus the properties of the monolayer
that contains this molecule can be engineered using recombinant
protein technology. Recombinant protein technology allows for
precise molecular engineering of proteins generated from
microorganisms such as bacteria and thus can be used to generate
oleosin species with different functionality and properties. To
demonstrate that this molecule has such modularity, green
fluorescent protein (GFP) mutant oleosin was expressed by fusing
eGFP to the N-terminus of the 42-30G-63 oleosin. The modified
oleosin genes are constructed using standard molecular biology
techniques and cloned into the expression vector pBamUK.
eGFP-functionalized oleosin is added to the aqueous phase during
microbubble generation. It is evident that the microbubbles
produced with the blend of the two oleosin species (pure at 1 mg
mL.sup.-1, mutant at 0.05 mg mL.sup.-1) along with 10 mg mL.sup.-1
(PEO).sub.78-(PPO).sub.30-(PEO).sub.78 has the eGFP mutant species
incorporated in the bubble shell, whereas the microbubbles
generated without the eGFP mutant species do not show any
fluorescence. FIGS. 13a-d, which confirm this finding, depicts
confocal fluorescent microscopy images of bubbles produced with (a
and b) oleosin and (c and d) with a blend containing the eGFP
mutant. Also fluorescence intensity was observed to be fairly
uniform on the surface of the bubbles with no signs of phase
separation, which has been observed on microbubbles that have been
stabilized with mixture of phospholipids. These results indicate
that oleosin with different functionalities can be generated and
incorporated into the microbubble shell and that oleosin
distributes uniformly on the surface of microbubbles.
Echogenicity measurements were carried out using microbubbles
generated with a solution containing 1 mg mL.sup.-1 oleosin and 10
mg mL.sup.-1 (PEO).sub.78-(PPO).sub.30-(PEO).sub.78. Microbubbles
were collected directly in a .about.3 cm long dialysis tubing with
a diameter of 16 mm, which was sealed at one end and pre-filled
with PBS solution containing 10 mg mL.sup.-1
(PEO).sub.78-(PPO).sub.30-(PEO).sub.78. Microbubbles were flown
directly into the dialysis tube from the PDMS device outlet using
polyethylene tubing, which was submerged in the PBS solution. After
collecting a desired amount of microbubbles, the tube was sealed on
the other end to avoid introducing any air pockets and was stored
in 50 mL centrifuge tubes filled with PBS solution containing 10 mg
mL.sup.-1 (PEO).sub.78-(PPO).sub.30-(PEO).sub.78. The tube was kept
on a spinning wheel rotating at 60 rpm to induce continuous motions
of the microbubbles and more importantly to remove large bubbles
that may have been collected. The echogenicity of these
microbubbles was tested using a broadband high-frequency ultrasound
transducer at 7-15 MHz in brightness mode (B-mode). The
microbubbles, with a radius of about 4 .mu.m are acoustically
active along the entire length of the dialysis tube as shown in
FIG. 14. In contrast, a PBS solution containing 10 mg mL.sup.-1
(PEO).sub.78-(PPO).sub.30-(PEO).sub.78 without any microbubbles
does not show any acoustic signal, indicating that the
oleosin-stabilized microbubbles are highly echogenic. Microbubbles
remain acoustically responsive 30 min after the initial measurement
and even one week after the first measurement, showing
non-detectable changes in the signal brightness. These results
indicate that these microbubbles stabilized with oleosin are highly
stable and echogenic and thus could have significant potential for
theranostic applications.
Example 5
Microbubbles Production and Characterization
The liquid phase containing the shell material included oleosin or
a solution containing oleosin proteins and
(PEO).sub.78-(PPO).sub.30-(PEO).sub.78 or
(PEO).sub.100-(PPO).sub.65-(PEO).sub.100 diluted in
phosphate-buffered saline (PBS) (pH 7.2, Sigma-Aldrich, St Luis,
Mo., USA). The components were mixed together to the desired
concentration. Microbubbles were generated using liquid phases
containing different combinations of the three components. The
liquid phase consisting of oleosin and
(PEO).sub.n-(PPO).sub.m-(PEO).sub.n triblock copolymers, at the
optimal concentration dispersed in PBS were supplied to the
microfluidic device using a Harvard Apparatus PHD Ultra syringe
pump (Harvard Apparatus, Holliston, Mass.) at flow rates between
500 .mu.L h.sup.-1 to 1000 .mu.L h.sup.-1. To connect the channels
to syringes, polyethylene tubing with an inner diameter of 0.38 mm
and an outer diameter of 1.09 mm (BB31695-PE/2, Scientific
Commodities Inc, Lake Havasu City, Ariz.) was used. The gas phase
having 99.999% pure nitrogen gas (N.sub.2, GTS Welco, Richmond,
Va.) or octafluorocyclobutane (C.sub.4F.sub.8) (SynQuest
Laboratories, Alachua, Fla.) was supplied to the device using a
pressure regulator (Type 700, ControlAir Inc., Amhrest, N.H.) at
pressures between 15 and 20 psi. Polyethylene tubing with an inner
diameter of 0.86 mm and an outer diameter of 1.32 mm (BB31695-PE/5,
Scientific Commodities Inc, Lake Havasu City, Ariz.) was used
connect the channel to the pressure regulator. The membrane valve
was actuated using a dual-valve pressure controller
(PCD-100PSIG-D-PCV10, Alicat Scientific, Tucson, Ariz.) at pressure
between 0 and 40 psi.
Microbubbles were produced by first applying a small pressure to
the gas inlet (2-4 psi) immediately followed by injecting the
liquid phase at the desired flow rate (500-1000 .mu.L h.sup.-1).
The gas phase was then increased slowly until steady state of
bubble generation is reached. Images of microbubbles production
were captured using an inverted microscope (Nikon Diaphot 300,
Melville, N.Y.) connected to a high speed Phantom V7 camera. For
microbubbles that remained stable during generation and collection,
long term stability was characterized by collecting microbubbles at
the air-water interface in 35 mm petri dishes, acquiring images
under a Carl Zeiss Axio Plan II upright microscope (Carl Zeiss
Microscopy, Thornwood, N.Y.) connected to a QImaging Retiga 2000R
camera. Microbubbles diameter variation over time was measured and
images are analyzed using ImageJ (v 1.47v, NIH, USA).
Example 6
Ultrasound Imaging
Microbubbles for ultrasonic imaging were collected and imaged
directly in 16 mm membrane dialysis bag, which was pre-filled with
buffer solution and sealed at one end. After a desire amount of
bubbles was collected, the tube was sealed at the other end
carefully avoiding formations of air pockets. The collected
microbubbles were imaged using a clinical ultrasound scanner HDI
5000 (Phillips/ATL, Bothell, Wash., USA) equipped with a broadband
high-frequency ultrasound transducer at 7-15 MHz. Grayscale B-mode
images were acquired with a mechanical index (MI) of 0.37 and 0.47
with focus between 0.5-1.5 cm and 1-2 cm, respectively. Time gain
compensation (TGC) is fixed throughout the experiments.
Example 7
Tuning the Mechanical Properties of Recombinant Protein-Stabilized
Microbubbles Using Triblock Copolymer Surfactants
In this example, the mechanical properties of microbubbles
stabilized with recombinant protein oleosin-30G were studied using
micropipette aspiration technique. FIG. 20 illustrates physical
properties of Oleosin 30G (MW=15,206 g/mol) and different
Pluronic.RTM. surfactants (F68 MW=8,400, F77 MW=6,600, P105
MW=6,500, L64 MW=2,900).
As shown in FIG. 15a, microbubbles were generated by PDMS Hole
Array Method, which is a new method for formulating air-filled
microbubbles with tens-of-micrometer-size in diameter. This method
has the most valuable merit of using a small amount of protein
solutions, which lowers damage and contamination of high value
biological samples. As shown in FIG. 15b, the average radius of the
microbubbles (R.sub.b,avg) was controlled by the PDMS hole
sizes.
As shown in FIGS. 16a-d, microbubbles were generated with different
amounts of Pluronic.RTM. F68 and/or Oleosin-30G. FIG. 16a shows
microbubbles with F68 at 1 mg/ml; FIG. 16b shows microbubbles with
Oleosin-30G at 1 mg/ml; FIG. 16c shows microbubbles with
Oleosin-30G at 1 mg/ml and F68 at 10 mg/ml; FIG. 16d shows
microbubbles with Oleosin-30G at 1 mg/ml and F68 at 20 mg/ml. FIG.
16e shows changes in the radius of microbubbles stabilized at
different compositions as a function of time after collection. The
oleosin-30G plays a critical role to generate and stabilize the
microbubbles.
FIG. 17a illustrates micropipette aspiration of
oleosin-30G-stabilized microbubbles with Pluronic.RTM. F68
(Oleosin-30G at 1 mg/ml+F68 at 10 mg/ml). Negative pressure was
needed to grab and hold microbubbles firmly with the
micropipette.
FIGS. 18a-e illustrate the effect of blending different
concentrations of a membrane sealing agent, Pluronic.RTM. F68, with
Oleosin-30G. Pluronic.RTM. F68 has been used as a cell membrane
sealing agent to protect cells against external shocks. The mean
bursting membrane tension and the mean elastic area compressibility
modulus of the cells increased with increasing amounts of
Pluronic.RTM. F68.
FIGS. 19a and 19b illustrate the effects on mechanical properties
of Oleosin-30G microbubbles by adding different kinds of
Pluronic.RTM. surfactants.
In this example, the real expansion modulus of the recombinant
protein-shelled microbubbles was controlled by blending different
types of triblock copolymer surfactants. The modulus of the
oleosin-30G microbubbles increased by blending a cell membrane
sealing agent, Pluronic.RTM. F68. The resulting a real expansion
modulus was dependent on the F68 concentration. Furthermore, it was
demonstrated that the Pluronic.RTM. triblock copolymers having
shorter hydrophilic chains, as compared to hydrophobic chains,
softened the oleosin-30G microbubble shells (see, e.g., FIG.
19b.)
Although the invention is illustrated and described herein with
reference to specific embodiments, the invention is not intended to
be limited to the details shown. Rather, various modifications may
be made in the details within the scope and range of equivalents of
the claims and without departing from the invention.
SEQUENCE LISTINGS
1
231140PRTArtificial SequenceOleosin Variant 1Gly Ser Ala Thr Thr
Thr Tyr Asp Arg His His Val Thr Thr Thr Gln1 5 10 15Pro Gln Tyr Arg
His Asp Gln His Thr Gly Asp Arg Leu Thr His Pro 20 25 30Gln Arg Gln
Gln Gln Gly Pro Ser Thr Gly Lys Leu Ala Leu Gly Ala 35 40 45Thr Pro
Leu Phe Gly Val Ile Gly Phe Ser Pro Val Ile Val Pro Ala 50 55 60Met
Gly Ile Ala Ile Gly Leu Ala Gly Val Thr Gly Phe Gln Arg Asp65 70 75
80Tyr Val Lys Gly Lys Leu Gln Asp Val Gly Glu Tyr Thr Gly Gln Lys
85 90 95Thr Lys Asp Leu Gly Gln Lys Ile Gln His Thr Ala His Glu Met
Gly 100 105 110Asp Gln Gly Gln Gly Gln Gly Gln Gly Gly Gly Lys Glu
Gly Arg Lys 115 120 125Glu Gly Gly Lys Leu Glu His His His His His
His 130 135 1402378PRTArtificial SequenceOleosin Variant 2Val Ser
Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val1 5 10 15Glu
Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu 20 25
30Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys
35 40 45Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr
Leu 50 55 60Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met
Lys Gln65 70 75 80His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr
Val Gln Glu Arg 85 90 95Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys
Thr Arg Ala Glu Val 100 105 110Lys Phe Glu Gly Asp Thr Leu Val Asn
Arg Ile Glu Leu Lys Gly Ile 115 120 125Asp Phe Lys Glu Asp Gly Asn
Ile Leu Gly His Lys Leu Glu Tyr Asn 130 135 140Tyr Asn Ser His Asn
Val Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly145 150 155 160Ile Lys
Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser Val 165 170
175Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro
180 185 190Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala
Leu Ser 195 200 205Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu
Leu Glu Phe Val 210 215 220Thr Ala Ala Gly Ile Thr Leu Gly Met Asp
Glu Leu Tyr Lys Gly Ser225 230 235 240Ala Thr Thr Thr Tyr Asp Arg
His His Val Thr Thr Thr Gln Pro Gln 245 250 255Tyr Arg His Asp Gln
His Thr Gly Asp Arg Leu Thr His Pro Gln Arg 260 265 270Gln Gln Gln
Gly Pro Ser Thr Gly Lys Leu Ala Leu Gly Ala Thr Pro 275 280 285Leu
Phe Gly Val Ile Gly Phe Ser Pro Val Ile Val Pro Ala Met Gly 290 295
300Ile Ala Ile Gly Leu Ala Gly Val Thr Gly Phe Gln Arg Asp Tyr
Val305 310 315 320Lys Gly Lys Leu Gln Asp Val Gly Glu Tyr Thr Gly
Gln Lys Thr Lys 325 330 335Asp Leu Gly Gln Lys Ile Gln His Thr Ala
His Glu Met Gly Asp Gln 340 345 350Gly Gln Gly Gln Gly Gln Gly Gly
Gly Lys Glu Gly Arg Lys Glu Gly 355 360 365Gly Lys Leu Glu His His
His His His His 370 3753192PRTArtificial SequenceOleosin Variant
3Met Ala Thr Thr Thr Tyr Asp Arg His His Val Thr Thr Thr Gln Pro1 5
10 15Gln Tyr Arg His Asp Gln His Thr Gly Asp Arg Leu Thr His Pro
Gln 20 25 30Arg Gln Gln Gln Gly Pro Ser Thr Gly Lys Ile Met Val Ile
Met Ala 35 40 45Leu Leu Pro Ile Thr Gly Ile Leu Phe Gly Leu Ala Gly
Ile Thr Leu 50 55 60Val Gly Thr Val Ile Gly Leu Ala Leu Ala Thr Pro
Leu Phe Val Ile65 70 75 80Phe Ser Pro Val Ile Val Pro Ala Met Ile
Ala Ile Gly Leu Ala Val 85 90 95Thr Gly Phe Leu Thr Ser Gly Thr Phe
Gly Leu Thr Gly Leu Ser Ser 100 105 110Leu Ser Tyr Leu Phe Asn Met
Val Arg Arg Ser Thr Met Ser Val Pro 115 120 125Val Gln Arg Asp Tyr
Val Lys Gly Lys Leu Gln Asp Val Gly Glu Tyr 130 135 140Thr Gly Gln
Lys Thr Lys Asp Leu Gly Gln Lys Ile Gln His Thr Ala145 150 155
160His Glu Met Gly Asp Gln Gly Gln Gly Gln Gly Gln Gly Gly Gly Lys
165 170 175Glu Gly Arg Lys Glu Gly Gly Lys Leu Glu His His His His
His His 180 185 1904192PRTArtificial SequenceOleosin
VariantMISC_FEATURE(43)..(129)Xaa at positions 43 to 129 may be any
naturally-occurring or artificial amino acid and up to 87 of them
may be absent 4Met Ala Thr Thr Thr Tyr Asp Arg His His Val Thr Thr
Thr Gln Pro1 5 10 15Gln Tyr Arg His Asp Gln His Thr Gly Asp Arg Leu
Thr His Pro Gln 20 25 30Arg Gln Gln Gln Gly Pro Ser Thr Gly Lys Xaa
Xaa Xaa Xaa Xaa Xaa 35 40 45Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa 50 55 60Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa65 70 75 80Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 85 90 95Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 100 105 110Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 115 120 125Xaa Gln
Arg Asp Tyr Val Lys Gly Lys Leu Gln Asp Val Gly Glu Tyr 130 135
140Thr Gly Gln Lys Thr Lys Asp Leu Gly Gln Lys Ile Gln His Thr
Ala145 150 155 160His Glu Met Gly Asp Gln Gly Gln Gly Gln Gly Gln
Gly Gly Gly Lys 165 170 175Glu Gly Arg Lys Glu Gly Gly Lys Leu Glu
His His His His His His 180 185 1905192PRTArtificial
SequenceOleosin VariantMISC_FEATURE(1)..(42)Xaa at positions 1 to
42 may be any naturally- occurring or artificial amino acid and up
to 42 of them may be absent 5Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 5 10 15Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Ile Met Val Ile Met Ala 35 40 45Leu Leu Pro Ile Thr Gly
Ile Leu Phe Gly Leu Ala Gly Ile Thr Leu 50 55 60Val Gly Thr Val Ile
Gly Leu Ala Leu Ala Thr Pro Leu Phe Val Ile65 70 75 80Phe Ser Pro
Val Ile Val Pro Ala Met Ile Ala Ile Gly Leu Ala Val 85 90 95Thr Gly
Phe Leu Thr Ser Gly Thr Phe Gly Leu Thr Gly Leu Ser Ser 100 105
110Leu Ser Tyr Leu Phe Asn Met Val Arg Arg Ser Thr Met Ser Val Pro
115 120 125Val Gln Arg Asp Tyr Val Lys Gly Lys Leu Gln Asp Val Gly
Glu Tyr 130 135 140Thr Gly Gln Lys Thr Lys Asp Leu Gly Gln Lys Ile
Gln His Thr Ala145 150 155 160His Glu Met Gly Asp Gln Gly Gln Gly
Gln Gly Gln Gly Gly Gly Lys 165 170 175Glu Gly Arg Lys Glu Gly Gly
Lys Leu Glu His His His His His His 180 185 1906192PRTArtificial
SequenceOleosin VariantMISC_FEATURE(130)..(192)Xaa at positions 130
to 192 may be any naturally-occurring or artificial amino acid and
up to 63 of them may be absent 6Met Ala Thr Thr Thr Tyr Asp Arg His
His Val Thr Thr Thr Gln Pro1 5 10 15Gln Tyr Arg His Asp Gln His Thr
Gly Asp Arg Leu Thr His Pro Gln 20 25 30Arg Gln Gln Gln Gly Pro Ser
Thr Gly Lys Ile Met Val Ile Met Ala 35 40 45Leu Leu Pro Ile Thr Gly
Ile Leu Phe Gly Leu Ala Gly Ile Thr Leu 50 55 60Val Gly Thr Val Ile
Gly Leu Ala Leu Ala Thr Pro Leu Phe Val Ile65 70 75 80Phe Ser Pro
Val Ile Val Pro Ala Met Ile Ala Ile Gly Leu Ala Val 85 90 95Thr Gly
Phe Leu Thr Ser Gly Thr Phe Gly Leu Thr Gly Leu Ser Ser 100 105
110Leu Ser Tyr Leu Phe Asn Met Val Arg Arg Ser Thr Met Ser Val Pro
115 120 125Val Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa 130 135 140Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa145 150 155 160Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa 165 170 175Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 180 185 1907192PRTArtificial
SequenceOleosin VariantMISC_FEATURE(1)..(129)Xaa at positions 1 to
129 may be any naturally- occurring or artificial amino acid and up
to 129 of them may be absent 7Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 5 10 15Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 35 40 45Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 50 55 60Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa65 70 75 80Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 85 90 95Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 100 105
110Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
115 120 125Xaa Gln Arg Asp Tyr Val Lys Gly Lys Leu Gln Asp Val Gly
Glu Tyr 130 135 140Thr Gly Gln Lys Thr Lys Asp Leu Gly Gln Lys Ile
Gln His Thr Ala145 150 155 160His Glu Met Gly Asp Gln Gly Gln Gly
Gln Gly Gln Gly Gly Gly Lys 165 170 175Glu Gly Arg Lys Glu Gly Gly
Lys Leu Glu His His His His His His 180 185 1908192PRTArtificial
SequenceOleosin VariantMISC_FEATURE(43)..(192)Xaa at positions 43
to 192 may be any naturally-occurring or artificial amino acid and
up to 150 of them may be absent 8Met Ala Thr Thr Thr Tyr Asp Arg
His His Val Thr Thr Thr Gln Pro1 5 10 15Gln Tyr Arg His Asp Gln His
Thr Gly Asp Arg Leu Thr His Pro Gln 20 25 30Arg Gln Gln Gln Gly Pro
Ser Thr Gly Lys Xaa Xaa Xaa Xaa Xaa Xaa 35 40 45Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 50 55 60Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa65 70 75 80Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 85 90 95Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 100 105
110Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
115 120 125Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa 130 135 140Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa145 150 155 160Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa 165 170 175Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 180 185 1909192PRTArtificial
SequenceOleosin VariantMISC_FEATURE(1)..(42)Xaa at positions 1 to
42 may be any naturally- occurring or artificial amino acid and up
to 42 of them may be absentMISC_FEATURE(130)..(192)Xaa at positions
130 to 192 may be any naturally-occurring or artificial amino acid
and up to 63 of them may be absent 9Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 5 10 15Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Ile Met Val Ile Met Ala 35 40 45Leu Leu Pro Ile Thr
Gly Ile Leu Phe Gly Leu Ala Gly Ile Thr Leu 50 55 60Val Gly Thr Val
Ile Gly Leu Ala Leu Ala Thr Pro Leu Phe Val Ile65 70 75 80Phe Ser
Pro Val Ile Val Pro Ala Met Ile Ala Ile Gly Leu Ala Val 85 90 95Thr
Gly Phe Leu Thr Ser Gly Thr Phe Gly Leu Thr Gly Leu Ser Ser 100 105
110Leu Ser Tyr Leu Phe Asn Met Val Arg Arg Ser Thr Met Ser Val Pro
115 120 125Val Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa 130 135 140Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa145 150 155 160Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa 165 170 175Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 180 185 19010134PRTArtificial
SequenceOleosin Variant 10Met Ala Thr Thr Thr Tyr Asp Arg His His
Val Thr Thr Thr Gln Pro1 5 10 15Gln Tyr Arg His Asp Gln His Thr Gly
Asp Arg Leu Thr His Pro Gln 20 25 30Arg Gln Gln Gln Gly Pro Ser Thr
Gly Lys Leu Ala Leu Ala Thr Pro 35 40 45Leu Phe Val Ile Phe Ser Pro
Val Ile Val Pro Ala Met Ile Ala Ile 50 55 60Gly Leu Ala Val Thr Gly
Phe Gln Arg Asp Tyr Val Lys Gly Lys Leu65 70 75 80Gln Asp Val Gly
Glu Tyr Thr Gly Gln Lys Thr Lys Asp Leu Gly Gln 85 90 95Lys Ile Gln
His Thr Ala His Glu Met Gly Asp Gln Gly Gln Gly Gln 100 105 110Gly
Gln Gly Gly Gly Lys Glu Gly Arg Lys Glu Gly Gly Lys Leu Glu 115 120
125His His His His His His 13011142PRTArtificial SequenceOleosin
Variant 11Gly Ser Glu Ala Thr Thr Thr Asn Asp Gln His His Val Thr
Thr Thr1 5 10 15Gln Pro Gln Asp Gln His Asp Gln His Thr Gly Asp Gln
Leu Thr His 20 25 30Pro Gln Asp Gln Gln Gln Gly Pro Ser Thr Gly Glu
Leu Ala Leu Gly 35 40 45Ala Thr Pro Leu Phe Gly Val Ile Gly Phe Ser
Pro Val Ile Val Pro 50 55 60Ala Met Gly Ile Ala Ile Gly Leu Ala Gly
Val Thr Gly Phe Gln Trp65 70 75 80Gln Asp Asn Val Asn Gly Glu Leu
Gln Asp Val Gly Glu Gln Thr Gly 85 90 95Gln Asn Thr Asn Asp Leu Gly
Gln Gln Ile Gln His Thr Ala His Glu 100 105 110Met Gly Asp Gln Gly
Gln Gly Gln Gly Gln Gly Gly Gly Asn Glu Gly 115 120 125Gln Asn Glu
Gly Gly Asn His His His His His His Asp Asp 130 135
14012140PRTArtificial SequenceOleosin Variant 12Gly Ser Ala Thr Thr
Thr Lys Asn Arg His His Val Thr Thr Thr Gln1 5 10 15Pro Gln Lys Arg
His Asn Gln His Thr Gly Asn Arg Leu Thr His Pro 20 25 30Gln Arg Gln
Gln Gln Gly Pro Ser Thr Gly Lys Leu Ala Leu Gly Ala 35 40 45Thr Pro
Leu Phe Gly Val Ile Gly Phe Ser Pro Val Ile Val Pro Ala 50 55 60Met
Gly Ile Ala Ile Gly Leu Ala Gly Val Thr Gly Phe Gln Trp Asn65 70 75
80Lys Val Lys Gly Lys Leu Gln Asn Val Gly Gln Lys Thr Gly Gln Lys
85 90 95Thr Lys Asn Leu Gly Gln Lys Ile Gln His Thr Ala His Gln Met
Gly 100 105
110Asn Gln Gly Gln Gly Gln Gly Gln Gly Gly Gly Lys Gln Gly Arg Lys
115 120 125Gln Gly Gly Lys Leu Glu His His His His His His 130 135
14013138PRTArtificial SequenceOleosin
VariantMISC_FEATURE(133)..(138)Xaa at positions 133 to 138 may be
any naturally-occurring or artificial amino acid and up to 6 of
them may be absent 13Gly Ser Thr Thr Thr Tyr Asp Arg His His Val
Thr Thr Thr Gln Pro1 5 10 15Gln Tyr Arg His Asp Gln His Thr Gly Asp
Arg Leu Thr His Pro Gln 20 25 30Arg Gln Gln Gln Gly Pro Ser Thr Gly
Lys Leu Ala Leu Ala Thr Pro 35 40 45Leu Phe Val Ile Phe Ser Pro Val
Ile Val Pro Ala Met Ile Ala Ile 50 55 60Gly Leu Ala Val Thr Gly Phe
Gln Arg Asp Tyr Val Lys Gly Lys Leu65 70 75 80Gln Asp Val Gly Glu
Tyr Thr Gly Gln Lys Thr Lys Asp Leu Gly Gln 85 90 95Lys Ile Gln His
Thr Ala His Glu Met Gly Asp Gln Gly Gln Gly Gln 100 105 110Gly Gln
Gly Gly Gly Lys Glu Gly Arg Lys Glu Gly Gly Lys His His 115 120
125His His His His Xaa Xaa Xaa Xaa Xaa Xaa 130 1351460PRTArtificial
SequenceHer-2 Affibody 14Val Asp Asn Lys Phe Asn Lys Glu Met Arg
Asn Ala Tyr Trp Glu Ile1 5 10 15Ala Leu Leu Pro Asn Leu Asn Asn Gln
Gln Lys Arg Ala Phe Ile Arg 20 25 30Ser Leu Tyr Asp Asp Pro Ser Gln
Ser Ala Asn Leu Leu Ala Glu Ala 35 40 45Lys Lys Leu Asn Asp Ala Gln
Ala Pro Lys Leu Glu 50 55 601531DNAArtificial SequencePrimer
15aaggagatag gatccaccac aacctacgac c 311628DNAArtificial
SequencePrimer 16gcaccgagag cgagcttgcc ggttgagg 281728DNAArtificial
SequencePrimer 17cctcaaccgg caagctcgct ctcggtgc 281834DNAArtificial
SequencePrimer 18ccttcacata atccctctga aacccggtaa cacc
341934DNAArtificial SequencePrimer 19ggtgttaccg ggtttcagag
ggattatgtg aagg 342033DNAArtificial SequencePrimer 20tatatgaatc
tcgagtttcc ccccttcttt tcg 3321105DNAArtificial SequencePrimer
21ctcgctctcg gtgcgactcc gctgtttggt gttataggtt tcagccctgt tattgttcca
60gcgatgggta tagcgattgg gcttgcgggt gttaccgggt ttcag
1052231DNAArtificial SequencePrimer 22atcggtatac atatggtgag
caagggcgag g 312328DNAArtificial SequencePrimer 23atctaaaatg
gatcccttgt acagctcg 28
* * * * *